Integrated crystal mounting and alignment system for high-throughput biological crystallography

ABSTRACT

A method and apparatus for the transportation, remote and unattended mounting, and visual alignment and monitoring of protein crystals for synchrotron generated x-ray diffraction analysis. The protein samples are maintained at liquid nitrogen temperatures at all times: during shipment, before mounting, mounting, alignment, data acquisition and following removal. The samples must additionally be stably aligned to within a few microns at a point in space. The ability to accurately perform these tasks remotely and automatically leads to a significant increase in sample throughput and reliability for high-volume protein characterization efforts. Since the protein samples are placed in a shipping-compatible layered stack of sample cassettes each holding many samples, a large number of samples can be shipped in a single cryogenic shipping container.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. patent application Ser. No.10/319,282 filed on Dec. 12, 2002, and entitled “Integrated CrystalMounting and Alignment System for High-throughput BiologicalCrystallography”, hereby incorporated by reference in its entirety,which in turn claims priority to Provisional Patent Application number60/341,020, filed on Dec. 12, 2001, and also entitled “IntegratedCrystal Mounting and Alignment System for High-throughput BiologicalCrystallography”, which is hereby incorporated by reference in itsentirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with U.S. Government support under ContractNumber DE-AC03-76SF00098 between the U.S. Department of Energy and TheRegents of the University of California for the management and operationof the Lawrence Berkeley National Laboratory. The U.S. Government hascertain rights in this invention.

REFERENCE TO A COMPUTER PROGRAM

Not applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to the transportation, roboticcrystal mounting and alignment, manipulation, mounting, alignment ofcrystal samples in a variety of experimental environments. The presentinvention more particularly relates to the mounting, alignment, andexposure of samples to synchrotron radiation for high-speed x-raycrystallography.

2. Description of the Relevant Art

Overview of X-ray Crystallographic Systems

Aspects of the present invention facilitate the transportation, as wellas the remote and unattended mounting and alignment of frozencrystals—of e.g. biological materials, such as proteins, lipids, ordeoxyribonucleic acids (DNA)—for x-ray diffraction analysis. A majorchallenge in the x-ray diffraction analysis system design is thenecessity of storing the samples in liquid nitrogen before mounting andfollowing removal, as well as maintaining the samples at near liquidnitrogen temperature throughout the mounting, alignment, and x-raydiffraction analysis data acquisition process. Additionally, theprecision and stability of the crystal sample location alignment must bevery high, with absolute sample position maintained within a few micronswhile rotating in one axis while being exposed to an incident x-ray beamthrough as much as a full 360°.

Traditional x-ray diffraction analysis crystal sample handlingprocedures are operator-intensive, requiring continuous manual operatorintervention at the measurement station. The ability to perform thesetasks remotely and automatically significantly increases crystalmounting and measurement throughput, as well as reliability forlarge-scale protein crystallography characterization. An increase inthroughput multiplies the number of samples that may be analyzed in agiven time period, thus decreasing the time per sample, thereby loweringthe cost associated with synchrotron-based x-ray crystallography.

Synchrotron-Based X-ray Crystallography

One embodiment of this invention is in the area of cryogenic proteincrystallography at synchrotron sources, although the robotic mountingand alignment system can be adapted for other laboratory x-ray sources.Potential uses include high-volume protein characterization experiments.The level of application of this invention could range from a smallexperimental program processing only a few samples per day to largeprojects screening and analyzing many thousands of samples per year.

Synchrotron-based x-ray crystallography is one application of thisinvention. Synchrotrons are capable of producing intense monochromaticpseudo-coherent photons of precisely controllable energies. The propertyof high intensity (otherwise known as high brightness) of thesynchrotron x-ray beams means that acquisition of crystal latticediffraction patterns can be done very rapidly, whereas other, lowerintensity beams may require several times longer for a diffractionpattern to be acquired. The high brightness of the synchrotronradiation, combined with the narrow energy bandwidth achievable using amonochromator, can lead to exceedingly high-resolution x-ray diffractionpatterns.

The x-ray diffraction patterns can subsequently be analyzed to infer therelative spatial positions of the atoms constituting the crystal latticestructure. The overall x-ray diffraction analysis of crystals is knownas x-ray crystallography. The information contained in the crystalstructure can lead to important insights about the function of themolecule and into molecular-chemical interactions. Such insights canlead to targeted, and thus faster, pharmaceutical development andimproved pharmaceuticals: a field known as ‘structure basedpharmaceutical design’.

Biological Crystallography Mounting Techniques

Currently, most x-ray crystallography work is done using synchrotronx-ray sources. These x-ray sources are extremely expensive to operate,which means that time is precious. However, since synchrotron x-raycrystallography is still a recent phenomenon, most sample mounting isdone manually, which is both slow and imprecise. Furthermore, sincecrystallography must be done on crystalline material, the sample must bemaintained in a frozen state. Typically, this is ensured by keeping thesample at near liquid nitrogen temperatures.

The requirement that the sample be maintained at liquid nitrogentemperature, however, requires that technicians and scientists can onlymount the samples using cumbersome techniques of indirectly handling thesample. Thus, people cannot be allowed to inadvertently heat the sample,and reciprocally, the sample handling tools, and sample handlingfixtures, cannot freeze the fingers of the people who do the mounting.To meet this requirement, clumsy tools resembling forceps or pliers areused. These tools are somewhat cumbersome, further adding time anddifficulty in mounting and handling the sample.

As more time is required to manually mount the sample, more heat istransferred from the ambient atmosphere, raising the crystal sampletemperature. Some biological crystal samples, frozen at a critical pointin a chemical reaction with another compound, continue their reactionsat temperatures as low at 100° K, only about 22° K above that of liquidnitrogen. This stringent maximum temperature requirement for somesamples implies that the sample must be actively cooled during theentire mounting process, which adds still further time and complexity tothe mounting process. It is preferable that the sample crystals becooled to a temperature not in excess of 150° K, more preferably not inexcess of 130° K, yet more preferably not in excess of 110° K, stillmore preferably not in excess of 100° K, yet still more preferably notin excess of 90° K, and most preferably not in excess of 80° K.

The largest time-related issue with manual operator mounting ofsynchrotron x-ray crystallography samples is that the humans must enterthe x-ray irradiation area (‘the hutch’) to mount and dismount thecrystal samples. This action involves in turn a sequence of safetyinterlocking steps to protect the personnel from a harmful andpotentially lethal dosage of x-rays used to irradiate the crystal sampleto generate the diffraction patterns. Typically, one or more heavylead-lined doors must be opened and closed, additional beam shuttersinserted, and interlocking safety devices must be carefully verified forsafe operation, prior to human access to the sample.

The result is that manual mounting of a synchrotron x-raycrystallography sample is slow. As a result of being slow, manualmounting is very expensive as measured in synchrotron beam time.

Biological Crystallography Sample Transportation

Currently, there are relatively few synchrotron x-ray sources availablefor x-ray crystallography. Therefore, scientists wishing to usesynchrotron x-ray sources face the dilemma of transporting the crystalsamples to the synchrotron while simultaneously maintaining thecrystal's cryogenically frozen state.

A particularly fruitful use of synchrotron x-ray crystallography is indetection of chemical interactions within a specific biological sample.These interactions are evanescent in nature, sometimes reacting in theone-nanosecond time scale. Additionally, typical biological processescan follow a number of biochemical pathways that are time dependent.Some of these biochemical pathways proceed even at temperatures as lowas 100° K. Thus, for a scientist to determine the crystalline structureof an intermediate state biochemical interaction, the biological samplemust be frozen to a temperature low enough to inhibit further reaction,typically close to the liquid nitrogen temperature of about 77° K undernormal laboratory conditions.

A Relevant Patent

Abbott Laboratories is the named assignee of U.S. Pat. No. 6,404,849 B1(the '849 patent), entitled “Automated Sample Handling for X-RayCrystallography”. The '849 patent discloses algorithms for centering acrystal at a reference position relative to home position sensors, aswell as the hardware for screwing a threaded sample holding device onand off a positioning device. The '849 patent uses a multi-axis robot tomove crystals from a sample rack to a positioning device.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed toward thetransportation and manipulation of samples of cryogenically frozenbiological particles, preferably protein crystals, mounted onstandardized base-pin configured sample assemblies.

The integrated crystal mounting and alignment system for high-throughputbiological crystallography which transports and manipulates the sampleassemblies comprises eight major components:

-   -   1) a sample repository having a storage Dewar filled with liquid        nitrogen, capable of keeping many samples cryogenically frozen,        with a sample repository stage able to addressably move the        sample assemblies to a point where a particular sample assembly        can be extracted;    -   2) a system for shipping, storing and handling of the sample        assemblies at cryogenic temperatures, preferably liquid nitrogen        temperatures;    -   3) a computer-controlled sampling system sequencing a particular        sample assembly through the steps of: a) selecting the        particular sample assembly from the cryogenic sample        repository, b) removing the selected sample assembly from the        sample repository, c) transferring the sample assembly to a        three axis positioner mounted on a goniometer head, d) centering        the sample in the x-ray beam, e) exposing the sample (held by        the mounted sample assembly) to x-ray radiation to produce a        crystallographic image at a sequence of rotational exposure        angles while simultaneously maintaining the sample's cryogenic        temperatures, and f) replacing the sample assembly back in the        cryogenic sample repository;    -   4) a sample gripper capable of firmly grasping a sample        assembly, while keeping the sample at cryogenic temperature;    -   5) a gripper stage, using the sample gripper to: remove the        sample assembly from cryogenic sample repository, transport the        sample to a sample positioner, and replace the sample assembly        in the sample repository, while at all times maintaining the        temperature of the sample at or below 78° K;    -   6) a sample gripper defroster capable of keeping the sample        gripper free of frost buildup during cycles of sample assembly        mounting and dismounting (or unmounting) in ambient humid air;    -   7) a sample positioner consisting of a precision three-axis        positioner mounted on a precision goniometer; and    -   8) an optical alignment system that provides feedback to the        sample positioner for precise alignment of the sample to a        predefined point in space within the x-ray beam during sample        rotation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross sectional view of a sample assembly with a crystalsample mounted.

FIG. 2A is a top view of a sample cassette with one sample assembly.

FIG. 2B is a cross sectional view through section 2-2 of FIG. 2A of asample cassette with one sample assembly.

FIG. 2C is an exploded view of the cross sectional view of FIG. 2B, witha sample cassette with one sample assembly.

FIG. 3A is a top view of a sample cassette cover.

FIG. 3B is a cross sectional view through section 3-3 of FIG. 3A of asample cassette cover.

FIG. 3C is a bottom view of a sample cassette cover showing liquidnitrogen venting features.

FIG. 4A is a cross sectional view of a sample cassette assemblycomprised of an assembled sample cassette with one sample assembly, andprotected by a sample cassette cover.

FIG. 4B is an exploded cross sectional view of a sample cassetteassembly comprised of a sample cassette, one sample assembly, and asample cassette cover, before assembly.

FIG. 5A is a cross sectional view of a sample cassette carrier with sixassembled sample cassettes present and the top slot vacant.

FIG. 5B is a bottom view of the sample cassette carrier and allassembled sample cassettes absent.

FIG. 6 is a cross sectional view of a cryogenic shipping container withsample cassette carrier with six sample cassette assemblies present, andthe top slot vacant.

FIG. 7A is a top view of a cassette deck with three sample cassettespresent and one sample cassette absent.

FIG. 7B is a sectional view of the cassette deck of FIG. 7A with threesample cassettes present and one sample cassette absent.

FIG. 8 is a cross sectional view of a sample gripper.

FIG. 9A is a partial front view of the integrated robotic crystalmounting and alignment system showing most of the major subsystems,where the system has just grasped a sample assembly in the samplerepository.

FIG. 9B is a partial front view of the integrated robotic crystalmounting and alignment system showing most of the major subsystems,where the system has retracted a pneumatic stage, known as SmallMove,causing the sample gripper to move vertically upwards.

FIG. 9C is a partial front view of the integrated robotic crystalmounting and alignment system showing most of the major subsystems,where the system has retracted the transverse vertical stage; known asUpDown, causing the sample gripper to move further vertically upwards.

FIG. 9D is a partial front view of the integrated robotic crystalmounting and alignment system showing most of the major subsystems,where the system has rotated the pneumatic 90° rotary stage, known asRotary, causing the sample gripper to rotate toward the samplepositioner.

FIG. 9E is a partial front view of the integrated robotic crystalmounting and alignment system showing most of the major subsystems,where the horizontal stage has moved the sample gripper in a longhorizontal translation, causing the sample gripper to move to apredetermined distance toward the sample positioner.

FIG. 9F is a partial front view of the integrated robotic crystalmounting and alignment system showing most of the major subsystems,where the SmallMove stage has moved the sample gripper a shorthorizontal translation to place the sample assembly in contact with themounting post of the sample positioner.

FIG. 9G is a partial front view of the integrated robotic crystalmounting and alignment system showing most of the major subsystems,where the horizontal stage has moved the sample gripper in a longhorizontal translation, causing the sample gripper to move to apredetermined distance away from the sample positioner, leaving a sampleassembly on the sample positioner mounting post.

FIG. 10A is a partial front view of the sample positioner, including atilt plate disposed between a goniometer and the X′ Y′ compound stage.

FIG. 10B is a partial front view of the sample positioner, includinganother tilt plate disposed between the X′ Y′ compound stage and the Z′stage.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT DEFINITIONS

“Biological crystal” means a crystallized frozen biological material,preferably a cryogenically frozen biological material at near liquidnitrogen temperatures.

“Biological material” means either a collection of independentmolecules, or a material having a non-covalently bound assembly ofmolecules derived from a living source. Examples include, but are notlimited to, complexes of proteins, lipoprotein particles comprised oflipoproteins and lipids; viral particles assembled from coat proteinsand glycoproteins; immune complexes assembled from antibodies and theircognate antigens, deoxyribonucleic acids (DNA), ribonucleic acids (RNA),polysaccharides, etc.

“Computer” means any device capable of performing the steps developed inthis invention to result in an optimal waterflood injection, includingbut not limited to: a microprocessor, a digital state machine, a fieldprogrammable gate array (FGPA), a digital signal processor, a collocatedintegrated memory system with microprocessor and analog or digitaloutput device, a distributed memory system with microprocessor andanalog or digital output device connected with digital or analog signalprotocols.

“Cryogenic” means a temperature at or below that of liquid nitrogen atstandard atmospheric pressure, −195.79° C. or 77.36° K.

“Degree of freedom” means any one of the ways that a mechanical systemcan change spatial configuration, examples include, but are not limitedto rotation, translation, and combinations of rotations and/ortranslations in one or more axes.

“Gage vacuum” means a relative pressure lower than ambient atmosphericpressure.

“Ferromagnetic” means a material capable of exhibiting alignment ofatomic or molecular magnetic domains. Such a material is capable ofmagnetization, and is subjected to forces in the presence of a magneticfield.

“Goniometer” typically means a device for measuring angles. Herein, itis used somewhat differently in that it rotates a surface to particularangles as instructed by a computer device using internal referenceangular measurements.

“Dewar” means any container for liquefied gases. These containers aretraditionally typically double-walled with an evacuated interior havinglow thermal emissivity surfaces to reduce heat transfer from thecontainer interior to the ambient atmosphere.

Overview

The present invention is directed toward the transportation andmanipulation of samples of cryogenically frozen biological materials,preferably protein crystals mounted on sample assemblies comprised of astandardized base-pin configuration.

When the hardware components are computer operated as a fully integratedcrystal mounting and alignment system for high-throughput biologicalcrystallography (herein referred to as the “system”), remote samplemounting and alignment of individual samples to a predetermined positionin space (for subsequent X-ray illumination) can be achieved in lessthan 30 seconds with minimal remote operator involvement. In someinstances, samples have been remotely mounted and aligned in a cycle of20 seconds. Since no human operator physical presence is required in theroom where active x-ray irradiation is taking place, there is noprolonged sequence of doors or interlocks to safely admit an operator,further increasing the speed of sample cycling.

The sample assembly used in this invention is comprised of a sample baseto which a thin sample tube is attached. The end of the sample tube notattached to the sample base has a sample loop, which contains the sampleof frozen biological material to be examined. Typical samples range from5-200 μm in size. The pin has a loop, which contains a cryogenicallyfrozen sample.

The system for shipping, storing and handling the samples is comprisedof a standardized sample cassette containing positions for a pluralityof sample assemblies, preferably 16 sample assemblies each in thepresent design. The outer dimension (diameter) of these sample cassettesis constrained by the maximum inner diameter of a standard cryogenictransport container. Presently, a preferred standard cryogenic transportcontainer will accommodate seven of these disks representing a maximumof 112 sample assemblies. Improvements on this design would allow evenmore sample assemblies in a higher packing density arrangement.

The computer-controlled high-throughput sample mounting and alignmentsystem has been designed as a device optimized for reliable removal ofthe samples from the liquid nitrogen sample repository system andmounting on a sample positioner. The sample positioner, as furtherdescribed below, positions the sample after it has been removed from thesample repository to a predetermined position in space through which anx-ray beam will subsequently pass, illuminating the sample and therebygenerating crystallographic diffraction patterns. The major subsystemsof the sample mounting and alignment system are: the sample positioner,a coordinated set of translational and rotating stages for positioningand orienting the sample gripper (known as the gripper stage), arepository system, a gripper defroster, an optical sample alignmentsystem, and an x-ray camera. This system positions a sample crystal at aprecise point in space for x-ray imaging analysis, or crystallography.

The sample repository system is principally a liquid nitrogen filledstorage Dewar removably placed atop a position-addressable repositorystage. It accommodates up to four sample cassettes on a cassette deckcorresponding to 64 samples per load. The individual sample assembliesare held fixed in the storage Dewar by a machinable magnetic materialthat attracts and retains the ferromagnetic material in the sampleassembly. The storage Dewar is mounted on a two axis movable platformthat provides alignment of any one of the 64 samples on the cassettedeck 700 (FIG. 7A) beneath the sample gripper (described below). Byusing a larger Dewar, more sample assemblies can be tested in oneloading. Loads of 300 or more sample assemblies may be used.

In the preferred embodiment, the repository stage has one rotationaldegree of freedom, and one linear degree of freedom. By using a rotarystage atop a linear stage, valuable real estate is conserved, as thestage never has to move more than half of the diameter of the(preferably axisymmetric) storage Dewar.

The storage Dewar itself has external position referencing features thatprecisely and removably locate the storage Dewar atop the rotary andlinear stages. The storage Dewar also has an internal referencing systemthat is aligned with the external position reference features. Aremovable cassette deck is placed through alignment pins in the internalreferencing system. In this fashion, by placing the storage Dewar on therepository stage, individual sample assemblies mounted on the cassettedeck can be moved to a specific location required for sample pickup andsubsequent mounting. Samples can likewise be moved from one position toanother (empty) position either on the same or different samplecassette.

The sample gripper contains a liquid nitrogen cooled split colletmechanism for grasping the sample assembly with sufficient force toovercome the magnetic attraction between the sample assembly base andthe sample cassette base with its magnetic material, as well as to breakfree of any ice binding between them. The sample gripper is designed toencapsulate the sample tube with the sample crystal-containing sampleloop within a liquid nitrogen temperature environment during the briefperiod when the gripper stage uses the sample gripper to transfer thesample assembly to the sample positioner.

The sample gripper contains a low thermal capacity (preferably very thinstainless steel) outer shroud, which provides a sheath-flow of warm drygas to prevent icing and frost formation during exposure to ambienttemperature, moisture-laden air. After the sample assembly is mounted,the sample is maintained in a cryostream of dry, near liquid nitrogentemperature gaseous nitrogen during subsequent crystallographicmeasurements. Sufficient cryostream flow is provided to maintain thesample at liquid nitrogen temperatures despite beam heating induced bythe probing synchrotron-produced x-ray heat load and closely juxtaposedambient atmosphere.

Removal of the sample assemblies from the sample positioner andreplacement in the sample repository is accomplished using the samesample gripper. After removal, the mounting post upon which the sampleassembly was just previously mounted could accumulate frost, interferingwith the magnetic retention of the next sample assembly. If this frostbecomes problematic, the mounting post could be actively heated.

The sample gripper is periodically warmed to remove any iceaccumulation. The gripper defroster provides either heated dry nitrogengas or heated dry air to the outer shroud of the gripper through apattern of small holes. Due to the low thermal mass of the outer shroudof the sample gripper (recall that it is preferably very thin stainlesssteel), defrost typically occurs in 6 seconds or less. The upper part ofthe sample gripper is furthermore heated, allowing the sample gripper tobe left in the liquid nitrogen Dewar indefinitely to keep it cold, butat the same time not damaging the pneumatic actuator mounted on topoperating at near room temperature, which cannot be exposed to cryogenictemperatures.

The gripper stage moves the sample gripper through the mechanicalmotions used for removal and placement of the sample assembly betweenthe sample positioning stage and the sample repository. The gripperstage comprises two orthogonal pneumatic translational motions (XY)(which could also be motor-controlled) upon which is mounted a thirdrotational (θ) motion mechanism. A relatively low force pneumaticactuator, mounted on the rotational motion mechanism, provides furtherindependent vertical movement. The low force pneumatic actuator preventsdamage from the sample gripper impacting misplaced sample assemblies orfrost.

The gripper stage motions are preferably based in part on pneumaticactuators to achieve precise and rapid motions in the liquid nitrogencooled environment. The three-degree of freedom, (XYθ) positioner of thegripper stage has been designed to move the sample gripper with therequired positioning precision within the restricted allowable physicalenvelope of the experimental setup. Electrically powered motors andactuators could potentially replace some or all of these pneumaticactuators, however, pneumatic actuators have proven to be simpler,cheaper, and easier to control and maintain in this application.

The sample positioner is a three-degree of freedom stage, mounted on aprecision goniometer to provide single axis rotation. The three-degreeof freedom stage has been custom designed to position a sample assemblymounted on a mounting post at a precise position in the synchrotronx-ray beam, and, in coordination with the goniometer, to rotate thesample about that point. The three degree of freedom stage consists oftwo orthogonal motor-controlled translational motions (XY) upon which ismounted a third Z axis motion mechanism. By coordinating the motions ofthe XY stage and the goniometer, a mounted sample can be rotated about adefined point in space despite initial eccentric mounting. The overallhysteresis of the three degree of freedom stage is about 1.5 micronswith a stability of about 1 micron. The system has been shown to besuperior to existing commercial versions with respect to positionprecision and stability.

The optical alignment system that provides feedback for precisealignment of the sample is based upon a high-resolution zooming camerathat is optically aligned to a point in space that will be subsequentlyilluminated by the synchrotron x-ray beam. A software reference pixellocation for the center of the x-ray beam is initially established.Visual images of the mounted crystal sample are then compared with thisreference. Any necessary positional corrections are calculated andexecuted by the sample positioner to position the sample over thesoftware reference position.

The software and user interface designs have been structured toaccommodate alternative centering feedback data based, for instance, onthe measured x-ray intensities or data quality as indicated by the x-raydata collection imaging device.

It should be noted that the system described herein could be used forpositioning cryogenically stored samples to a particular point in spacefor many alternative types of measurements. These measurements may bedone by observing the sample at a single prescribed point in space, orby observing the sample at a variety of rotational angles as it isrotated by the goniometer. Depending on the relative time durations ofthe sampling measurement, the sample may be statically paused at eachmeasurement, or may be continually rotated. An example of a continualrotation measurement could be that of short pulse-width laserillumination using short pulses having microsecond, nanosecond, orpicosecond pulse-widths. With such short pulse-widths, even a movingsample appears as if it were stationary.

Data collection may be used on any experimental device outputtinginformation that can be used as a measurement. Examples include, but arenot limited to: x-ray crystallography, photonics-based tomography,photonics-based diffraction, surface spectroscopy, fluorescencecorrelation spectroscopy, photon arrival time interval distributionanalysis, fluorescence resonance energy transfer methods, massspectrometry, and evanescent wave methods, scanning probe microscopy,taccimetry, profilometry, and atomic force microscopy.

Sample Assembly

Referring now to FIG. 1, a sample assembly 100 is comprised of a sample110 captured in a sample loop 120. The sample 110 is preferably frozenat cryogenic temperatures, such as that of liquid nitrogen at standardatmospheric pressure, −195.79° C. or 77.36° K. The sample loop 120 is inturn attached by adhesive 130, preferably alpha cyanoacrylate, to sampletube 140. The sample tube 140 is attached to sample base 160 byinsertion into hole 150. The sample base 160 has an upper land 180 forretention, and a lower recess 170 for mounting.

Sample base 160 is ferromagnetic, preferably easily free-machining ANSI1118 steel with zinc electroplating after machining for corrosionresistance. The sample tube 140 is preferably stainless steel or otherlow thermal conductivity material, preferably already containing anattached 50 to 1000 μm nylon sample loop 120.

Sample Cassette

Referring now to FIGS. 2A, 2B and 2C, a sample cassette 200 holding onesample assembly 100 is shown. The sample cassette 200 has a cassettebase 210 into which a keyed shaft 220 is affixed on one end 225. Amagnet 250, preferably a machinable magnet of polymeric matrix material,is inserted into the cassette base 210, preferably in a recess 255 inthe cassette base 210. The machinable magnet 250 has one opening 230aligned with each corresponding cassette base opening 265. Sampleassemblies 100 are then able to be loosely inserted into cassette base210 through openings 265 and be retained by the upper surface 260 ofmagnet 250, with cooling liquid nitrogen able to flow through eachmagnet 250 opening 230 and cassette base 210 opening 265 to directlycontact, and hence, cool the sample assembly 100 through direct contactwith lower recess 170 (shown in FIG. 1).

In this embodiment of the sample cassette 200, 16 sample assemblies canbe accommodated in a regular pattern forming two concentric circles.However, any other regularized or nonregularized pattern (not shown inFIG. 2B) could be used as well.

Keyed shaft 220 is present to align a cassette cover 300 (describedbelow) so that it does not damage the samples 110 when they are coveredfor transport or storage. Many other functionally equivalent methods,using multiple unkeyed shafts in a pattern, or external jigs, would alsoachieve the result that cassette cover 300 placement onto the samplecassette 200 would not damage any of the samples 110. The attachment ofkeyed shaft 220 is preferably by a threaded screw into a threaded recessin the keyed shaft, however, many other mechanically equivalentattachment methods would also work, including, but not limited to pressfit, shrink fit, adhesive attachment, welding, or threading.

Sample Cassette Cover

Referring now to FIGS. 3A, 3B and 3C, a sample cassette cover 300 is acylindrical shape with a keyed opening 310 passing through a threadedsection 330. A cylindrical neck 340 portion protrudes above the main topsurface 320, and is centered on the sample cassette cover 300 center305. An indexing feature 350 serves as clearance for an alignmentfeature to be discussed below.

In the sample cassette cover 300 bottom surface 355, pluralities ofsample assembly recesses 360 provide protection to samples assemblies100 (shown in FIG. 1) placed in them. In this embodiment, an outer ventring 370 and an inner vent ring 380 vent the sample assembly recesses360; these details are not shown with hidden lines in FIG. 3A tominimize confusion of hidden lines. The outer vent ring 370 and an innervent ring 380 are respectively ported to the exterior of the samplecassette cover 300 with pluralities of outer vent ports 375 and innervent ports 385. This venting arrangement allows for the flow of liquidnitrogen to each of the sample assembly recesses 360, ensuring thatsample assemblies 100 (shown in FIG. 1) as well as the cassette cover300 are amply cooled by liquid nitrogen. It also allows the venting ofnitrogen gas, which might otherwise build up inside the sample assemblyrecesses 360.

Sample Cassette Assembly

Referring now to FIG. 4A, a sample cassette assembly 400 is showncomprised of a sample cassette cover 300 assembled in place over asample assembly 100, and retained by sample cassette 200. Now referringto both FIGS. 4A and 4B, sample assembly 100 is magnetically retained onthe upper surface 260 of magnet 250.

During installation, the sample cassette cover 300 first encounterskeyed shaft 220. The sample cassette cover 300 must first berotationally aligned relative to sample cassette 200 so that the keyedshaft 220 may translate into the keyed opening 310. Since the keyedshaft 220 is taller than the installed sample assembly 100, the sample110 cannot be damaged by the sample cassette cover 300 during normalinstallation of the sample cassette cover 300 as sample assemblyrecesses 360 in the sample cassette cover 300 slides over each of thesample assemblies 100.

The sample cassette cover 300 has one sample assembly recess 360 foreach matching cassette base 210 opening 265. The depth of the sampleassembly recesses 360 exceeds the retained height of the sample assembly100. Additionally, the sample assembly recesses 360 have smallerdiameters than the width of the sample assembly 100, so that the bottomsurface 355 of the sample cassette cover 300 positively retains thesample base 160 by contacting upper land 180.

When assembled as shown in FIG. 4A, the sample assemblies 100 arepositively sandwiched between the sample cassette cover 300 and thesample cassette 200. Now referring additionally to FIG. 3C, the outervent ring 370 and the inner vent ring 380 allow for filling of thesample assembly recesses 360 with liquid nitrogen through outer ventports 375 and inner vent ports 385.

In one embodiment of the invention, the assembled cassette cover 300 andsample cassette 200 are inverted so that sample assembly recesses 360form liquid nitrogen repositories, thus keeping the biological crystalsample 110 immersed in liquid nitrogen and maintaining the sample at acryogenic temperature until all of the liquid nitrogen has boiled away.In this embodiment, several minutes of room temperature exposure can betolerated by the sample with minimal temperature rise when movingsamples from shipping container to cryogenic sample repository.

Additional mechanical components (not shown) clip and retain the samplecassette cover 300 to the sample cassette 200, although many othermethods of positively retaining the parts together exist, and arereadily designed by those skilled in the mechanical design arts.

Sample Cassette Carrier

Refer now to FIGS. 5A and 5B, where a cassette carrier 500 is depicted.A top hook 510 is press fit into a low thermal conductivity sleeve 515,preferably comprised of fiberglass or other low thermal conductivity lowtemperature plastic, which has inserted into it a stainless steel rod520. The stainless steel rod 520 is welded to a tab 525 formed by twonarrow slots 530 on either side. The tab 525 is part of a sheet 580 thatencompasses, and is attached to, about half the diameter of a pluralityof shelves 550. The method of attachment could be any that survivesrepeated thermal shocks from room temperature to liquid nitrogentemperature. In this embodiment, three screws 590 are used to attacheach shelf 550 to the sheet 580.

The distances between the shelves 550 form a set of shelf openings, orlandings 535. For purposes of illustration, the top-most landing 535 isvacant, without a sample cassette assembly 400. The other six shelveopenings in the diagram each show sample cassette assemblies 400present.

In FIG. 5B, each shelf 550 has an inner 551 and outer 552 radiusconcentric about a center point 555, which is the same center point fora mounted sample cassette assembly 400. During insertion of the samplecassette assembly 400, cylindrical neck 340 (shown in FIGS. 3A and 3B)of the sample cassette cover 300 slides into obround slot 560, whereretaining spring 570, secured by fastener 575, retains the samplecassette cover 300. Retaining spring 570 deflects partially intoretaining spring recess 565. When fully inserted, cassette cover 300center 305 is roughly concentric with center point 555.

Cryogenic Transport Container

Before shipping, the cryogenic transport container 600 of FIG. 6 isinitially precooled with liquid nitrogen for shipping per themanufacturer's directions. Some of these precooling steps can take aslong as four hours to complete.

Referring now to FIG. 6, cryogenic transportation container 600 has aremovable insulation plug 610, which inserts into a correspondingcylindrical bore 630 in bulk insulation 620. When cylindrical bore 630is initially empty except for nitrogen gas and liquid, the cassettecarrier 500, having one or more sample cassette assemblies 400, is firstinserted. Subsequently, the removable insulation plug 610 is installed.Further liquid nitrogen, if needed, is added per the manufacturer'sdirections. Depending on the manufacturer of cryogenic transportationcontainer 600, ambient temperature exposure, and the detailedconstruction of the container, cryogenic temperatures below −150° C. canbe maintained during shipment for up to 200 hours.

Cassette Deck

Refer now to FIGS. 7A and 7B. The cassette deck 700 is shown. Thecassette deck 700 has a center reference hole 710, three mounting holes760, and a larger diameter keying hole 750 to uniquely orient thecassette deck 700 with respect to hardware incorporated into the samplerepository (not shown). Orientation pins 720 are preferably press fitinto the cassette deck 700. The orientation pins 720 provide a uniqueorientation of sample cassettes 200 which are positioned between the twoother outer positioning pins 740. This pattern is replicated in fourquadrants. In one quadrant there is just an outline of the area 745 thatis normally occupied by a sample cassette 200.

By using this arrangement, in conjunction with the sample cassette 200design, all sample assemblies 100 are uniquely positioned with respectto the cassette deck 700. The unique positioning allows for unattendedsample assembly 100 mounting and demounting using a sample gripperdescribed below.

Sample Gripper

The sample gripper 800 is shown in FIG. 8. An upper actuator flange 865is connected to a lower actuator flange 805. The actual mechanism of theactuator is not shown, as these are readily commercially available aseither solenoidal electrical or pneumatic force/displacement devices.The preferred actuator is pneumatic. The lower actuator flange 805 hasbeen modified so that in conjunction with gripper flange 815, a port 810is formed. The port 810 attaches to a plenum 812. The plenum 812 allowsa continuous gas connection with a series of cylindrical openings 820,which at their apex, connect to small openings 825. Input gas can beattached to port 810, fill plenum 812, pass through a plurality ofcylindrical opening 820, and emit at small openings 825 into an outershroud area 832. The outer shroud area 832 is formed by an inner tube830 and outer tube 835, which are both attached to gripper flange 815.The outer tube 835 necks down to a removable close fitting shroud tube840. The inner tube 830 has a very low thermal mass, low heat capacitymaterial, and is preferably both very thin walled, and made of stainlesssteel. At the lower end of the inner tube 830, is attached a colletsleeve 845. The collet sleeve 845 is preferably silver soldered (notshown) to the stainless steel inner tube 830. The inner tube 830 must besufficiently thick so as to keep from bucking under axial compressiveforces generated by the collet sleeve 845.

The split collet 850 has an actuation movement relative to the colletsleeve 845, closing the split collet 850 about a sample assembly 100located within its grasp. When split collet 850 is retracted upwards,the collet sleeve 845 causes compressive closure of the collet actuationsurface 846, with consequent high force retention of the sample assembly100 located within the split collet 850 in a collet recess 847. Thesplit collet 850 is pulled upward by collet adapter 854, which connectsthe split collet 850 to the collet tube 855. The collet tube 855 is inturn connected to the actuator adapter 860. The actuator adapter 860connects collet tube 855 to the actuator tube 870. Thus a verticalmotion of the actuator tube 870 causes the same vertical motion of theactuator adaptor 860, collet tube 855, collet adapter 854, and in turnthe split collet 850.

The temperature of the split collet 850 is measured by a temperaturesensing element 852 located at the bottom of a temperature sensing hole851. Wires (not shown to minimize drawing clutter) ascend upward throughthe temperature sensing hole 851, through a matching hole in colletadapter 854, and exit the sample gripper 800 through the center bore 875of actuator tube 870.

To cool the sample gripper 800 down to temperatures appropriate forsample assembly 100 pickup (e.g. liquid nitrogen temperature), thecollet sleeve 845 end of the sample gripper 800 is immersed in liquidnitrogen. At this time, there is no sample assembly 100 present. A smallgage vacuum of 3-4 inches of mercury is drawn on port 810, which iscommunicated through the small openings 825 to the outer shroud area832. Vent port 833 in inner tube 830 allows the vacuum to pull liquidnitrogen up and around split collet 850. Since the collet is split,liquid nitrogen fills the interior 853 of the split collet 850. Thetemperature-sensing element 852 is used to register when the splitcollet 850 temperature has cooled sufficiently for sample assembly 100pickup.

When the sample gripper 800 is moving the sample assembly 100, roomtemperature dry nitrogen gas is fed through port 810 into the outershroud area 832 to preclude frost buildup on inner tube 830 or splitcollet 850. The frost buildup is prevented by the simple expedient ofkeeping moisture away from any of the cold surfaces of the inner tube830 or split collet 850, by the flow of the dry nitrogen gas, preferablyin laminar flow.

The sample gripper 800 is drawn showing most details, with the centersection abbreviated by a cut 890.

Integrated Crystal Mounting and Alignment System for High-ThroughputBiological Crystallography

Referring now to FIG. 9A, we see the integrated crystal mounting andalignment system for high-throughput biological crystallography 900 asviewed down an axis parallel to the incoming synchrotron x-ray beam 901.A frame 902 connects the various subsystems, and will not be fullydescribed other than to say that it must be sufficiently stable andstiff to keep most components accurately positioned to within about 1μm.

The subsystems include a repository stage, a gripper stage, a samplepositioner, a cryostream unit, a video alignment subsystem, and acollimation and beam blocking subsystem. These subsystems are more fullydescribed sequentially below.

The repository stage is comprised of the Y₁ linear stage 904, which ismounted on the frame 902. Atop the Y₁ linear stage 904 is attachedrotary stage 906, which rotates about a vertical axis of revolution.Storage Dewar 908 removably attaches to the rotary stage 906 at arepeatable position and orientation using standard mechanical andprecision engineering fixturing techniques that are well known in thesearts. The storage Dewar 908 is nominally filled with enough liquidnitrogen 910 to amply cover any sample assemblies 100 that may bepresent in any sample cassettes 200. The cassette deck 700 is mounted onposition referencing components not described here, which allows samplecassettes 200 to be addressably positioned relative to the frame 902with high accuracy.

The gripper stage moves the sample gripper 800 relative to the frame902. It comprises a horizontal stage 926, an UpDown stage 924, and arotary stage 920. A long travel Y₂ linear stage, known as the horizontalstage 926, moves horizontally (along the plane of the paper) a verticalstage mounted transversely thereon, known as UpDown 924. The UpDownstage 924 comprises a platform 922 that serves as a mounting base for a90° rotary stage 920, known as Rotary, preferably a pneumatic 90° rotarystage. The 90° rotary stage 920 top mounts a small travel, lighteractuation force pneumatic stage called SmallMove 918, which serves as amount for the sample gripper 800. Rotary stage 920 rotate the samplegripper 800 between a downward position (as shown in FIG. 9C) and ahorizontal position (as shown in FIG. 9D).

The sample positioner is comprised of a high precision rotary stageknown as a goniometer 928, which is mounted on the frame 902. Thegoniometer 928 rotates an angle θ (theta) along an axis typicallyparallel to the horizontal plane. Upon the goniometer 928 is mounted a.A Z′ stage 932 with a magnetic mounting post 934 mounts onto the X′Y′stage 930. At the particular goniometer 928 angle θ depicted in FIGS.9A-9G, the X′Y′ stage 930 moves in and out of the plane of the paper(the X′ axis), and up and down in the plane of the paper (the Y′ axis).These motions will rotate with continued rotations of the goniometer 928in a typical kinematic rotating frame of reference. The mounting post934 is mounted upon, and moved by, compound X′ Y′ stage 930. Themounting post 934 is spring preloaded (to prevent hard sample assembly100 mountings), and rides on two sets of three bearings, each set ofwhich forms an equilateral triangle.

In an alternate embodiment of the system, the sample positioner isfurther comprised of a tilt plate 929 (as depicted in FIG. 10A) disposedbetween the high precision rotary stage known as the goniometer 928, andthe compound X′ Y′ stage 930, to which the Z′ stage 932 is attached asbefore. The effect of the tilt plate 929 is to rotate the Z′ stage 932eccentrically with respect to the axis of rotation of the goniometer 928so that the positioner rotates the sample 110 about an axisnon-orthogonal with the compound X′ Y′ stage 930 and the Z′ stage 932.The tilt plate 929 preferably forms a tilt angle of at least 15°, morepreferably of at least 10°, yet more preferably of at least 5°, stillmore preferably of at least 2°, and most preferably of at least 1°. Thealignment and centering operations described below may be used eitherdirectly by ignoring the effect of the tilt plate 929, or by includingthe angle of the tilt plate 929 in the alignment and centeringalgorithms. Regardless of the eccentricity induced by the tilt plate929, the properly centered sample 110 will maintain location within thex-ray synchrotron beam 901 when the beam is operational, and the samespatial position when the x-ray synchrotron beam 901 is non-operational,as further described below. Rotations of the sample 110 will normallyrequire operation of the compound X′ Y′ stage 930 and the Z′ stage 932for correct positioning.

In yet another embodiment (shown in FIG. 10B) another tilt plate 931 maybe disposed between the compound X′ Y′ stage 930 and the Z′ stage 932 toeffect the eccentricity described above. In this further embodiment, theother tilt plate 931 would preferably form a tilt angle of at least 15°,more preferably of at least 10°, yet more preferably of at least 5°,still more preferably of at least 2°, and most preferably of at least1°.

In operation, a sample assembly 100 (already removed here, and thus notshown) is retained by the mounting post 934 by magnetic attraction. Themounting post 934 could readily be heated to prevent frost formation,but heating has not yet proven necessary. The coordinated motions of therotation addressable goniometer 928, the compound X′ Y′ stage 930 andthe Z′ stage 932 allow a sample to be rotated in space about apredetermined point, preferably the incoming synchrotron x-ray beam 901.

A commercially available cryostream unit 936 emits a stream of nearliquid nitrogen temperature nitrogen gas to cool the sample 110 when thesample assembly 100 is mounted on the mounting post 934. The cryostreamunit 936 is actuated along axis 938 so as to: 1) prevent interferencewith the sample gripper 800 when mounting or unmounting sampleassemblies 100 (not shown), 2) not optically occlude the camera 948optical aperture, and 3) not interfere with the projection of theincoming synchrotron x-ray beam 901, regardless of whether or not thex-ray beam 901 is operational.

The video alignment subsystem is comprised of a commercially availablebacklighter 956 on a vertically extendible backlighter stage 958. Duringalignment, the backlighter stage 958 raises the backlighter 956 so thatthe sample 100 (not shown, but mounted on mounting post 934) is back-litwhen viewed by the high resolution macroscopic zooming video camera 948.During x-ray irradiation, the backlighter 956 is retracted so that it isout of the direct x-ray beam 901.

Collimation and beam blocking is typically required to respectively forma parallel incoming x-ray beam of a controlled diameter, or stop thebeam altogether. For collimation to work properly, a small aperture mustbe aligned with the incoming x-ray beam. Collimation and beam blockingof the x-ray beam is effected by using the collimator vertical actuator940 to raise a piezoelectric actuator 942, to which an x piezoelectricactuator 944 is attached, which moves a selection of collimators havingvarious diameters and beam blocks 946 into the x-ray beam 901. Note thatthe collimators and beam blocks 946 with their associated actuators, arein a non-interfering plane from the backlighter 956 so that each mayoperate independently without collision.

To collimate or locally block the synchrotron x-ray beam 901, thevarious diameter collimators and beam block 946 is moved up into thex-ray beam 901 by the collimator vertical actuator 940, and is preciselypositioned for optimal collimation by small, precise movements effectedby piezoelectric actuators 942 and 944.

Application of the Invention to Mount a Sample Assembly

Refer now to FIGS. 9A-9F, which is a sequence of partial front views ofthe integrated crystal mounting and alignment system for high-throughputbiological crystallography 900 with most of the major subsystemsillustrated. The sequence of FIGS. 9A-9F show some of the major stepsinvolved in conveying a sample assembly 100 to the sample positionermounting post 934 for alignment using camera 948 and subsequent datacollection from crystallographic diffraction of the incoming synchrotronx-ray beam 901 by the sample 110 (not shown).

Initially, the sample gripper 800 must be cooled sufficiently to safelygrasp a sample assembly 100. The sample gripper 800 is initiallypartially immersed in the liquid nitrogen 910 so that thetemperature-sensing element 852 (shown in FIG. 8) is cooled to atemperature of at least −150° C. prior to continuing with the sampleassembly pickup. This initial sample gripper 800 immersion is locatedaway from any resident sample assemblies 100 present in the storageDewar 908. For this purpose the sample gripper 800 is immersed in thestorage Dewar 908 until a set temperature is achieved. This initialcooling procedure typically requires over a minute. However, in normaloperation, the cooling down procedure is only required once in a set ofsamples since the sample gripper 800 remains cold with repeatedimmersions in the liquid nitrogen 910. When a sample assembly 100 pickupoccurs, further additional cooling will take place as the sample gripper800 is immersed in the storage Dewar 908.

FIGS. 9A-G correspond to the integrated crystal mounting and alignmentsystem 900 moving through a sequence of configurations as described morefully below. It is appreciated that there are many alternative sequencesand minor variations that may be used to effect the same operations.

In FIG. 9A, the system is in the process of using the sample gripper 800to grasp a sample assembly 100 in the storage Dewar 908. From there, itwill move the sample assembly 100 to the sample positioner mounting post934 for alignment and x-ray probing. At this step in the protocol, asample assembly 100 has been grasped by sample gripper 800. Prior tograsping the sample assembly 100, the following setup steps haveoccurred: (1) the gripper 800 is released; (2) the heater 950,collimator beam blocks 946, the cryostream unit 936 are retracted so asto not interfere with other movements; (3) Rotary 920 is rotated down sothat the sample gripper 800 assumes a vertical orientation; (4) UpDown924 has been moved down; (5) SmallMove 918 has been downwardly extended;and (6) the gripper stage has moved the sample gripper 800 to a locationin the storage Dewar 908, and immersed the sample gripper 800 in theliquid nitrogen 910 until the sample gripper 800 has reached atemperature of −130° C. as measured by the temperature sensing element852 (indicated but not shown in FIG. 8). Gripping is accomplished bymoving the sample gripper 800 over a selected sample assembly 100located in a predefined location pattern in the storage Dewar 908. Thesample gripper 800 is actuated, causing the split collet 850 to exert apressure on the sample assembly 100. The friction generated by thispressure is sufficient to overcome the frost buildup and/or magneticattraction of the sample assembly 100 to the sample cassettes 200 in thestorage Dewar 908.

Next, in FIG. 9B, the system has retracted the SmallMove 918 pneumaticstage, causing the sample gripper 800 to be moved vertically upwards.Depending on the liquid nitrogen 910 fill level in the storage Dewar908, the sample assembly 100 may have cleared the liquid nitrogen 910surface, as depicted here. Not shown in the drawings is an automatedliquid nitrogen fill apparatus, to keep the liquid nitrogen 910 filllevel at a specified level. The storage Dewar 908 is typically nearlyfull, so that the sample gripper 800 is partially immersed even whenSmallMove 918 is retracted to its highest vertical position.

Next, in FIG. 9C, the system has actuated the transverse vertical stage,known as UpDown 924, causing the platform 922 to move further verticallyupwards, carrying the sample gripper 800 vertically upwards, so that thegrasped sample assembly 100 vertically clears the top of the storageDewar 908.

Next, in FIG. 9D, the system has rotated the pneumatic 90° rotary stage920, known as Rotary, causing the sample gripper 800 and sample assembly100 to rotate 90° clockwise and point toward the sample positionermounting post 934. Sample assembly 100 is essentially collinear withmounting post 934.

Next, in FIG. 9E, the sample gripper 800 has moved by the longhorizontal travel linear stage, known as the Y₂ stage 926, causing thesample gripper 800 to move to a predetermined distance toward the samplepositioner mounting post 934, in a vector parallel to the Z′ stage 932motion (horizontally as indicated in FIG. 9E), with sample assembly 100approaching the mounting post 934 in advance of sample gripper 800.

Next, in FIG. 9F, the SmallMove 918 pneumatic stage has been extended ina short horizontal translation to mount the sample assembly 100 gentlyin contact with the magnetically attractive mounting post 934 of thesample positioner. After contact with the mounting post 934 isaccomplished, the gripper assembly 800 releases its grip on the sampleassembly 100. Now the cryostream unit 936 is actuated along line 938 toapproach the sample 110 mounted on the sample assembly 100 mounted onthe mounting post 934. The cryostream unit 936 is then activated to coolthe sample assembly 100 sample 110 (still cryogenically shielded in thegripper assembly 800) which is now roughly located at a spatial positionwhere the x-ray beam 901 will subsequently irradiate.

Next, in FIG. 9G, the sample gripper 800 has been moved a predetermineddistance away from the sample positioner mounting post 934 by the longtravel Y₂ linear stage, known as the horizontal stage 926. Since thesample gripper 800 has already been released, the sample assembly 100remains on the sample positioner mounting post 934 due to magneticattraction. Since the cryostream unit 936 has already been activated,the sample 110 is released from the sample gripper 800 cryogenicinterior directly into the cold stream of the cryostream unit 936. Inthis manner, the sample 110 is never exposed to ambient roomtemperature.

Initial Sample Reference Position Setup

In this system, a zooming microscopic camera 948 views providesinformation to correctly position the mounted sample 110 (shown earlierin FIG. 1). In order to establish this spatial position, athree-dimensional position in space must be aligned with the incomingsynchrotron x-ray beam 901 while the beam is active. With the x-ray beam901 active, a pin, or other small axisymmetric alignment shape is movedinto the beam until the beam is partially occluded. An x-ray imagingcamera, operationally similar to the high resolution macroscopic zoomingvideo camera 948, except that x-rays are detected, and zooming is notlikely necessary, is used to image the axisymmetric alignment shape.Either the synchrotron beam current must be greatly reduced, or morepreferably the x-ray beam intensity is greatly attenuated by anattenuator to prevent burnout of the camera for this operation.

With the x-ray beam 901 producing an image on the x-ray imaging camera,the axisymmetric alignment shape (typically a small bead, or pin with apoint) is moved by coordinated movements of the X′ and Y′ compound stage930, and Z′ stage 932 axes. Eventually, the movements are manually (orpossibly computer controlled) coordinated until the alignment shapeenters the field of view of the x-ray imaging camera. By coordinatedmovements of the X′ Y′ compound stage 930, and Z′ stage 932, androtation of the goniometer 928, a three-dimensional reference locationfor the center of the x-ray beam relative to the X′ Y′ compound stage930, and Z′ stage 932 is developed. Once the alignment shape is alignedto the x-ray beam, the beam may be turned off, or blocked completely, asit is no longer necessary for the initial alignment, as the x-ray beamcenter relative to the X′ Y′ compound stage 930, and Z′ stage 932 isalready known.

The backlighter stage 958 now raises the backlighter 956 so that thealignment shape is back-lit when viewed by the high resolutionmacroscopic zooming video camera 948. Since the x-ray beam may now beturned off, it is safe for personnel to enter the potentially irradiatedhutch area to manually (or by remote control of appropriate tilt orpointing actuators) align the camera 948 to view the alignment shape.Once the camera 948 is correctly positioned to view the alignment shapein roughly the center of field of view, no further camera 948 alignmentshould be necessary. The camera 948 is then used to view the alignmentshape. The alignment shape is viewed, and the pixel locationcorresponding to the portion of the alignment shape previouslypositioned in the center of the x-ray beam is recorded. This is thepixel location of the beam center at a particular zoom magnification.The zoom magnification is then increased to a higher magnification so asto more completely fill the field of view of the camera 948, and thepixel location again corresponding to the portion of the alignment shapepreviously positioned in the center of the x-ray beam is recorded.

Note that the zooming camera 948, can typically only determine positionin two dimensions as imaged pixel locations. Typical imaging devices canonly focus within a particular optical depth of field, which, dependingon the depth of field of the optical image, can provide additionalinformation regarding a distance from the optical objective by eitherbeing in or out of focus. In this instance, the zooming camera 948 ispreferably parfocally focused on the alignment shape; so that it remainsin focus at all zoom magnifications.

Note that, at this time, there are two reference positions being used:the software pixel location of the sample as viewed by the zoomingcamera 948, which is a two dimensional pixel reference position relatedto the field of view of the zooming camera 948; and the spatial centerof the alignment shape relative to the positioner, a three dimensionalreference. These software pixel locations, and the spatial position ofthe alignment shape relative to the positioner which has previously becollocated through the center of the x-ray beam, are subsequently usedto rapidly align samples for x-ray crystallography.

Sample Position Setup

Once the sample assembly 100 has been positioned on the positioner asdepicted in FIG. 9G, the sample 110 must be aligned to be concentricwith the x-ray beam 901 when it is activated. The previously obtainedsoftware pixel locations (two dimensional information) and the spatialcenter of the alignment shape relative to the positioner (threedimensional information) are used to correctly center the sample crystal110 for x-ray crystallography. These coordinates are cooperatively usedto position the sample crystal 110 relative to the positioner so thatthe sample crystal 110 may be rotated about the point where the x-raybeam 901 passes when it is activated.

In this system, a zooming microscopic camera 948 initially views thesample 100 (shown earlier in FIG. 1) at minimum magnification, andproduces video images of the mounted sample crystal 110. The images areread by a video frame-grabber to provide a digital image of the sample.By either manual or computer algorithmic operation, the frame pixelcoordinates of the center of the sample may be determined. The samplepositioner (comprised of X′ Y′ compound stage 930, Z′ stage 932, and thegoniometer 928) is then actuated to translate the sample 110 to thesoftware reference pixel location arrived at during initial zoomingcamera 948 alignment in a plane roughly (preferably within 45°, morepreferably within 30°, yet more preferably within 15°, and mostpreferably within 5°) parallel to the image plane of the camera 948. Thesample positioner then rotates the sample 110 through angular movementof the goniometer 928, and the process is repeated. At each rotation,the translational increments of the X′ and Y′ compound stage 930, and Z′stage 932 axes are recorded. Subsequently, these coordinates are used toarrive at the true three-dimensional spatial center of the sample 110crystal relative to the positioner. The entire process is thenoptionally repeated at higher zoom magnification levels as necessary.This sample 110 spatial center may be determined relative to thepositioner in as few as two rotations due to the short depth of field ofthe camera 948 at maximum zoom.

The distance from the alignment shape center to the sample 110 centerforms an offset vector. The offset vector is used to coordinate themovement of the sample 110 by relative movements of the X′ Y′ compoundstage 930, and Z′ stage 932 at each rotation. In this manner, the sample110 may be rotated in space through a point collocated with the centerof the x-ray beam 901 when it is operated. The x-ray beam 901 is notallowed to strike the sample 110 during alignment, so as to minimize anysynchrotron-produced x-ray 901 heating or x-ray induced chemicaldegradation.

Sample X-ray Crystallography

In the normal operation of the system, the sample positioner is moved sothat the sample 110 is positioned to a location where synchrotrongenerated x-rays 901 will be emitted after sample 110 alignment. Afterthe sample 110 is aligned as described above, the synchrotron x-ray beam901 is unblocked, allowing x-rays to irradiate the sample 110, which canthen be rotated to any arbitrary angular position while remainingcentered within the x-rays beam 901. After x-ray crystallography iscomplete, the synchrotron x-ray 901 source is again blocked or shutteredso as to interrupt delivery of the x-ray beam, effectively turning offthe x-ray beam. This blocking and unblocking of the x-ray source isimportant since the x-rays can induce damage to the crystalline sample,thereby degrading the data collected.

Sample Dismounting

Following the FIGS. 9G-9A in reverse is essentially the sequence ofmotions used by the unmounting protocol, where the sample assembly 100initially mounted on the sample positioner mounting post 934 is finallyreplaced in the storage Dewar 908.

For the remaining sample assemblies 100, the sequence of stepspreviously described is performed in reverse, from 9G (the present datacollection state), to 9F, 9E, 9D, 9C, 9B, and 9A where the sampleassembly 100 is replace in the Dewar 908. The sample gripper 800 isretracted sufficiently to clear the sample assemblies 100, but stillpartially immersed in the liquid nitrogen 910. The Y₁ linear stage 904and rotary stage 906 are actuated to position the next sample assembly100 beneath the sample gripper 800. At this point, the process repeatsfor sampling of the remaining sample assemblies 100.

After a period of use, the sample gripper 800 may become frost covered.A warm up or defrost protocol is used to remove any accumulated frostfrom the sample gripper 800. Although the spatial configuration fordefrosting is not directly shown in any Figure, it is readilyvisualized. During the defrost cycle, a heater 950 is extended by anIn/Out stage 952, and warm dry nitrogen gas is emitted from the heater950 onto the sample gripper 800, which has previously been moved intoposition for defrosting.

Conclusion

All publications, patents, and patent applications mentioned in thisapplication are herein incorporated by reference to the same extent asif each individual publication or patent application were eachspecifically and individually indicated to be incorporated by reference.

The description given here, and best modes of operation of theinvention, are not intended to limit the scope of the invention. Manymodifications, alternative constructions, and equivalents may beemployed without departing from the scope and spirit of the invention.In particular, the sequence of motions used in mounting and demountingsample assemblies 100 may be re-sequenced in a myriad of permutationswithout deviating from the general goal to be achieved so long ascomponents and subsystems do not destructively interfere with eachother.

1-20. (canceled)
 21. A method of high-throughput sample mounting andaligning comprising the steps of: a. selecting and removing a sampleassembly by a sample gripper from a sample repository pattern bymovement of the sample gripper and a gripper stage, where a sample ismounted on the sample assembly; b. mounting the sample assembly on thesample positioner by movement of the gripper stage and the samplegripper; c. addressably positioning the sample to a predetermined pointin space by movement of the sample positioner; d. rotating the samplethe sample essentially about the predetermined point in space for datacollection by movement of the sample positioner; and e. dismounting thesample assembly by moving the sample gripper back to the samplerepository pattern by movement of the sample gripper and the gripperstage; wherein:
 1. the sample positioner is attached to a frame;
 2. thesample gripper is attached to the frame by the gripper stage; and
 3. thesample repository has a plurality of the sample assemblies arrayed in apattern, and the sample repository is mounted to the frame by a samplerepository stage.
 22. The method of positioning a sample at apredetermined point in space of claim 21 further comprising:illuminating the sample with x-ray beam photons, and imaging adiffraction resulting from the illumination step to produce adiffraction data image at a particular rotation, the diffraction dataimage stored on a computer.
 23. The method of positioning a sample at apredetermined point in space of claim 22 further comprising: imaging ofthe sample at a plurality of rotations to produce a plurality ofdiffraction data images stored on a computer at each rotational angle.24. The method of positioning a sample at a predetermined point in spaceof claim 23 further comprising: ceasing the illumination of the samplewith x-ray beam photons after the plurality of diffraction data imagesstored on a computer at each rotational angle has been completed, andcomputing a crystal composition and atomic placement from the pluralityof diffraction data images.
 25. The method of positioning a sample at apredetermined point in space of claim 23 further comprising: cooling thesample at all times to a temperature not in excess of 150° K.
 26. Themethod of positioning a sample at a predetermined point in space ofclaim 23 further comprising: cooling the sample at all times to atemperature not in excess of 80° K.
 27. A method of computer-controlledimaging of a particular sample assembly comprising the steps of: a.selecting a particular sample assembly from a cryogenic samplerepository, b. removing the selected sample assembly from the samplerepository, c. transferring the sample assembly to a three axispositioner mounted on a goniometer head, d. centering a sample mountedon the sample assembly at a software reference pixel location, e.exposing the centered sample to x-ray radiation to produce acrystallographic image at a sequence of rotational exposure angles whilesimultaneously maintaining the sample's cryogenic temperatures, and f.replacing the sample assembly back in the cryogenic sample repository.28. The method of computer-controlled imaging of a particular sampleassembly of claim 27 wherein said centering step comprises: a. centeringthe sample within the depth of field of the software reference pixellocation.
 29. The method of computer-controlled imaging of a particularsample assembly of claim 27 wherein said centering step comprises: a.centering the sample by backlighting the sample.
 30. The method ofcomputer-controlled imaging of a particular sample assembly of claim 27wherein said removing step comprises: a. grasping the sample assemblywith a sample gripper, while b. keeping the sample at cryogenictemperature.
 31. The method of computer-controlled imaging of aparticular sample assembly of claim 30 wherein said keeping the sampleat cryogenic temperature step comprises: a. keeping the sample at orbelow 78° K.
 32. The method of computer-controlled imaging of aparticular sample assembly of claim 30 wherein said keeping the sampleat cryogenic temperature step comprises: a. pulling liquid nitrogen upand around a split collet in the sample gripper.
 33. The method ofcomputer-controlled imaging of a particular sample assembly of claim 27wherein said centering step comprises: a. measuring x-ray intensities asindicated by an x-ray data collection imaging device.
 34. The method ofcomputer-controlled imaging of a particular sample assembly of claim 27wherein said centering step comprises: a. measuring data quality asindicated by an x-ray data collection imaging device.
 35. A method ofhigh-throughput sample mounting and aligning comprising the steps of: a.means for selecting and removing a sample assembly, where a sample ismounted on the sample assembly; b. means for mounting the sampleassembly on a sample positioner; c. means for positioning the sample toa predetermined point in space by movement of the sample positioner; d.rotating the sample the sample essentially about the predetermined pointin space for data collection by movement of the sample positioner; ande. using said selecting and removing means for replacing the sampleassembly.
 36. The method of positioning a sample at a predeterminedpoint in space of claim 35 further comprising: illuminating the samplewith x-ray beam photons, and imaging a diffraction resulting from theillumination step to produce a diffraction data image at a particularrotation, the diffraction data image stored on a computer.
 37. Themethod of positioning a sample at a predetermined point in space ofclaim 22 further comprising: imaging of the sample at a plurality ofrotations to produce a plurality of diffraction data images stored on acomputer at each rotational angle.
 38. The method of positioning asample at a predetermined point in space of claim 23 further comprising:ceasing the illumination of the sample with x-ray beam photons after theplurality of diffraction data images stored on a computer at eachrotational angle has been completed, and computing a crystal compositionand atomic placement from the plurality of diffraction data images. 39.The method of positioning a sample at a predetermined point in space ofclaim 23 further comprising: cooling the sample at all times to atemperature not in excess of 150° K.
 40. The method of positioning asample at a predetermined point in space of claim 23 further comprising:cooling the sample at all times to a temperature not in excess of 80° K.